Recovery and concentration of bacitracin



United States Patent RECOVERY AND CONCENTRATION OF BACITRACIN LouisChaiet, Newark, and Thomas J. Cochrane, Jr.,

Rahway, N.J., assignors to Merck & Co., Inc., Rahway, N.J., acorporation of New Jersey N 0 Drawing. Application October 18, 1955Serial No. 541,308

3 Claims. (Cl. 167-65) 7 to 100 cc. of water.

solution thereof, such as from a fermentation broth. The

having been originally introduced into the broth as a nutrient media.

Bacitracin is an antibiotic many of the properties of which aredescribed in US. Patent No. 2,498,165 as well as some methods for itsproduction. Those methods involve somewhat tedious solvent anddistillation techniques, require relatively large volumes of expensivesolvents and produce relatively low yields.

Recovery and concentration of bacitracin from broths containingsynthetic nutrient media has been proposed by adsorption on, and elutionfrom, a zeolitic material (such as bentonite, magnesium trisili'cate,fullers earth and diatornaceous earth). This is not a practical processwhen working with a broth containing a significant quantity ofproteinaceous material, as described above.

Other methods have been proposed for the recovery and concentration ofbacitracin, but they have various objectionable features, such as one ormore of the following: multiplicity and complexity of steps, the needfor handling relatively large volumes of solutions (some of them beingrelatively expensive items), and inability to work satisfactorily withbroth having a relatively high content of proteinaceous material.

It is an object of this invention to provide an improved method ofrecovering and purifying bacitracin from aqueous solutions containingthis antibiotic. Another object is to provide an improved method ofpurifying a relatively concentrated aqueous solution of bacitracin.Other objects will be apparent from the detailed description hereinafterset forth.

In accordance with the present invention, it has been found thatbacitracin may be simply, effectively and inexpensively recovered andconcentrated from solutions thereof by adsorbing the bacitracin on asynthetic organic cation exchange resin on the hydrogen. cycle, and theneluting the bacitracin from the resin with a base. This was unexpected,since the bacitracin is an essentially neutral antibiotic and issomewhat unstable at a pH greater than about 9.0.

In the preferred, and most efficient, form of this invention which isparticularly applicable to the recovery and concentration of bacitracinfrom fermentation broth containing a substantial amount of proteinaceousmaterial such as soy bean meal, the cation exchange resin has a lowcross linkage, of the order of 2%.

It is also preferred that the elutriant be a weak base. Dilute aqueousammonia is particularly preferred, as it produces a salt-freeconcentrate when the eluate is concentrated by heat under vacuum.

The following examples illustrate specific embodiments Patented Dec. 1,1959 of the invention, but it is understood that such examples are notto be considered as limiting the invention.

EXAMPLE 1 The starting material was 2 liters of crude or wholebacitracin broth, assaying 77.6 units/ml. by microbial assay and havinga pH of about 8.2. This broth was the end product of the deepfermentation of a strain of B. subtilis in an aqueous nutrient mediumcontaining essentially soy bean meal in the ratio of 6 grams of soy beanmeal (solvent extracted type, for reasons of economy) The solids contentof the fermentation media at the start of the fermentation cycle wasthus 6%, practically all of proteinaceous material. At the end of thecycle the solids content was about 3 to 4%, so this was the solidscontent of the whole broth used as the starting material of thisexample.

The 2 liter sample of the whole broth, having a total activity of155,000 units, was acidified to pH 2 with concentrated sulfuric acid(about 8.3 ml.). Supercel (200 gms.) was added, and the solutionfiltered, thus removing the mycelia. The filter cake was washed with 400ml. of water, and the two filtrates were combined, giving a volume of2,100 ml. assaying 67.2 unit/ml., for a total activity of 141,000 units.

The pH of this filtered broth was adjusted to 4 by adding 10% sodiumhydroxide (using approximately 43 ml.) and then the filtered broth waspassed downflow through a column of 92 ml. of Duolite C-25 ion exchangeresin having a low (2%) cross linkage. This was a cation exchange resinavailable from the Chemical Process Company of Redwood City, California,and prepared from a matrix produced by the copolymerization of styrenewith divinylbenzene. Further details of this resin are described in thepaper presented to the 128th meeting of the American Chemical Society onSeptember 14, 1955, by IrvingM. Abrams, of the Chemical Process Co., andabstracted in Abstracts of Papers, 128th meeting of the AmericanChemical Society, Minneapolis, Minn, page 25M. The amount of crosslinkage involved is, by accepted practice, defined in terms of percentof divinylbenzene which is used in preparing the resin. Sixteen percentis a highly cross linked resin, while about 5 to 8% is the amount ofcross linking that is more or less standard. Hence it is evident thatthe 2% cross linkage present in the resin used in this example is asignificantly low cross linkage.

The resin used was in the form of porous beads having as its functionalgroups nuclear sulfonic acids. The resin was operated on the hydrogencycle, and the column had an inside diameter of 3.5 cms.

The filtered broth was passed through this resin column at the rate of3.1 m1./minute (i.e. 30 minute superficial contact time). The resincolumn was washed downflow with ml. of water to displace the broth fromthe column, and then the column was backwashed with water until theeffiuent was clear. The eflluent from the passage of the broth throughthe column, plus the effluents from the downflow wash and backwash, werecombined and are for convenience referred to as spent broth. The volumeof this spent broth was 2,620 ml. and assayed 1.2 units/ ml., giving atotal of approximately 3,100 units. Hence the resin removed 138,000units of bacitracin from the 141,000 units of filtered broth. passedthrough it, or 98% of the bacitracin activity.

The resin column was then eluted with, 1 N ammonium hydroxide at therate of 1.84 mL/minute (i.e. 50 minute superficial contact time). Therich cut was collected starting from the time the effiuent began tochange from a clear to a colored liquid. This was about the time asample of the effluent, when tested with a picric acid test solution,would produce a precipitate, indicating that the efiiuent containedbacitracin activity in a concentration exceeding 3 units/ml. Thecollection of the rich cut was stopped when the efiiuent becamecolorless or only very slightly colored. This is about the As the assaysfor bacitracin activity were made by microbial methods, and as suchmethods are recognized as having a possibility of error of up to plus orminus 10%, this is evidently the reason why the total activity point atwhich the picric acid test of a sample of the 5 of the concentrated richcut of batch No. 1 in the above effluent fails to produce a precipitate.table is reported as exceeding by about 5% the total The picric acidtest solution was prepared by stirring activity of the rich cut beforeit was concentrated. about 1 gm.hof plicric acid in 500 ml. of waterfgr'thirtg Till: tvgo effluents froirli the secongl resin colutlrninIINCI'G minutes. T e cear picric acid so ution was ecante corn inegiving a tota volume 0 610 ml. w ic asand used as the test solution. Inmaking the test, 2 ml. sayed 322 units/ml., so that the solution had atotal of of the test solution were added to 2 ml. of the bacitracin196,000 units of bacitracin activity. This solution was solutioncontaining 1 drop of glacial acidic acid, plus concentrated by heatingunder vacuum until the volume 1 or more additional drops of glacialacetic acid, dewas 95 ml., and then defecated by adding 5.8 m1. of apendent upon the amount of ammonium hydroxide conaqueous solution ofaluminum sulfate tained in the bacitracinsolution. Turbidity in thebaci- 15 (A1 (S0 18H 0) tracin solution indicated the presence of morethan 3 2 4 2 units of bacitracin per milliliter. and 0.9 gram ofsupercel and then adjusting the pH to The rich cut collected was 212milliliters having a pI -I 7.5 by adding sodium hydroxide solution.After filterof around 10 to 11 and assayed 498 units/mL, so that it 2ing the solution to remove the precipitate, the filtrate was contained atotal of 160,000 units of act vity. This was 0 144 ml. assaying 1430units/ml., so that the total ac- 77% of the activity adsorbed on theresin and 76% of tivity of the filtrate was 206,000 units of bacitracin.tll'ile act vity present in the filtered broth passed through v Toproduce zinc bacitracin from this filtrate, the pH t e resin. wasadjusted to about 4.4 by adding sulfuric acid, then A tail cut of 59 ml.of effluent was collected. This 5 ml. of 10% aqueous solution of zincchloride was gs slgyged 6 3 unit's/ml so that it contained a total ofonly added, followed by adjusting the pH to 7.5 by adding units 0acitracin activity. I sodium hydroxide. The product, formed as aprecipi- Promptly upon completion of the rich cut, it was placed tate inthe solution, was removed by filtering, and the in an evaporator operateunder vacuum so as'to drive 'filter cake dried under vacuum, yielding1.9 grams of off excess ammonia and concentrate the solution. This driedzinc bacitracin having an activity of 76 units per "confeiitrationswasterrlili nlate d whlen thle pH of the conmilligram, or a total activityof 144,000 units.

cen ra e was at w ic time t e vo ume was 188 ml. The over-all yield wasthus 144,000 units from 283,000 assaying 588 units/Ink S t t t e tOactivity of t e units in the starting material, representing 51%. Theconcentratewas 111,000 units of bacitracin. yield across the resincolumn was for batch No. 1, 106,000 While this wasa highly concentratedsolut on of baciunits from 141,000 units introduced into the column,tracin, t was desirable to remove, or reduce to negligible representing75%, and for batch No. 2, 91,800 units proportion, various impuritiesthat were present in this from 118,000 units introduced into the column,represolutlon before proceeding to a dried finished product. senting78%. The decrease in the volume of the baci- The concentrated rich cutfrom the resin column was tracin solution (across the resin column), wasfrom therefore passed through a column of 92 ml. of Duolite 2100 ml. to212 ml. in batch No. l, and from 1970 ml. P;Zhion exchanges Z6811;/(operatin(g on the acetate cycle) to 190 ml. in batch No. 2,representing the elimination a e ra e o m. minute Le. 50 minute superof90% of the initial volume for each batch. At the ficial contact time).Th s was an ion exchange resin same time, the potency:of the solutionwas increased avalllcilibig from the Chem cal Process Company and is a7.4 fold for batch N0. 1, and 7.7 fold for batch No. 2. wea ly asiiiiacid adsorbi ng resin in the form of porous In the steps whereconcentration was effected by heatgranu es an having as its functionalgroups primary, ing and also where the product was dried, theseoperawslegsohiiecllary tililnj6tertliari amtines. T'tllhe resin columnwas tions were performed under vacuum and at temperatures wi m o wa er.e tota eflluent was a below 45 C. purified gggicentrated solution ofbacitracin having a vOl- The first resin column was regenerated upon thecomllme 0 uf P 0f and ying 396 units/ml. pletion of each elution cyclebefore passing another batch so that thesolution had a total of 90,300units of baciof broth through the column. This was done by washl i fiactlvltying the column downfiow with water, then passing a 5 e aboveprocess was repeated for a second batch percent aqueous solution ofsodium hydroxide through the of similar whole bacitracin broth, using aregenerated column downfiow, followed by a water wash, and then firstresin column and an unregenerated second resin by a 5 percent solutionof sulfuric acid downfiow to colililmn. The following table compares theresults at return the resin to the hydrogen cycle. The column was 686 P-water washed downfiow until the pH of the efiluent rose Table I 4 BatchNo. 1 Batch No. 2 Material Vol., Assay, Total V 1., A '1 ml. u./ml.Activity, pH nil. uj iiii. Actii i t y, pH

units units 1. st ti t ri l h 2. tallestiiti i--.f .iiffi fi-- 3; $38333 E2888 .5 fill 23 iii'888 First Column 3. Spent broth 1.2 pg r iuuato 2 4 102.533 'i33 it? 1333 3:33;: 6. Concentratddiihh Cut 188 ass11%008 "5.7 180 is 50o 9.0

Second Column 7. Eflluent 228 396 90, 300 4.6 Not separately measured toabove 3.0, and then the column was backwashed with water, after whichthe resin was allowed to settle and the water drained off from thecolumn to just above the top of the resin bed.

The second resin column was regenerated upon the completion of eachsecond batch passed through it. This was done by passing a aqueoussolution of sodium hydroxide through the column downflow, followed by awater wash, downflow, until the pH of the efiluent dropped to below11.0. Then a 5% aqueous solution of acetic acid was passed through thecolumn, downflow, followed by a water wash, downflow, until the pH ofthe efiluent rose to above 4.0. The column was thenbackwashed withwater, after which the resin was allowed to settle and the water drainedoff from the, column to just above the top of the resin bed.

EXAMPLE 2 Two liters of bacitracin whole broth similar to that used inExample 1, and assaying 85 units/ml., was acidified to pH 3 withsulfuric acid and filtered with supercel admix. The filtered broth waspassed downflow through a column containing 75 ml. of Duolite (3-25 2%crosslinked resin on the hydrogen cycle at a rate of 4 ml./ minute,followed by 150 ml. of water as a wash. The bacitracin was eluted fromthe resin with l N ammonium hydroxide. The rich cut, of 130 ml., wasthen neutralized with sulfuric acid. The following table shows theresults:

The rich cut was then purified by solvent extraction techniques, asfollows: The pH of the solution was adjusted to 7.2 by adding ammoniumhydroxide, then 26 ml. of n-butanol was added. and the mixture stirredso that much of the bacitracin activity was extracted from the waterinto the butanol, while much of the impurities remained in the aqueoussolution. After settling, the butanol layer was removed, and the processrepeated four times using 13 ml. of butanol each time. The combinedbutanol extracts had a volume of 77 ml. and assayed 1520 units/ml. andcontained a total of 117,000 units of activity.

The rafiinate (i.e. water solution), after evaporation to 105 ml.,assayed only 35 units/ml. so that it contained only 3650 units ofactivity; I t

The bacitracin was recovered from the butanol by removing the butanol byazeotropic distillation with Water under vacuum, with the maximumtemperature about 35-40 C. The resulting aqueous solution of bacitracinwas 90 ml., assaying 1380 units/ml., for a total activity of 124,000units.

This solution of bacitracin was further purified by adding 5 ml. of. 20%aqueous solution of aluminum sulfate (Al (SO .18H O) and 1 gram ofsupercel, then adjusting the pH to 7.5 by adding sodium hydroxidesolution, and finally filtering the solution to remove the precipitate.The filter cake was washed with Water, and the combined filtrate was 150ml. assaying 690 units/ml., for a total activity of 103,000 units.

The filtrate, after cooling for 20 hours in the refrigerator, wasconcentrated to 90 ml. by heating under vacuum. The bacitracin in thefiltrate was converted to zinc bacitracin by adding 2.2 ml. of aqueoussolution of zinc chloride, following by adjusting the pH of the solutionm7 and stirring for 30 minutes. The solution was then filtered and thefilter cake, after washing, was dried under vacuum, yielding 1.21 gramsof dried zinc bacitracin having an activity of 80 units per milligram,fora total ac tivity of 97,000 units; The over-all yield wasthus 97,000units from 170,000 units in the starting material, representing 57%. Theyield across the resin. column was 125,000 units from 172,000 unitsintroduced into the column, representing'78%. The decrease in the volumeof the bacitracin solution across the resin column), was from 2460 ml.to 130 ml., representing the elimination of 95% of the initial volume,while the potency of the solution was increased 13.8 fold.

EXAMPLE 3 I downflow through a column containing 92 ml. of AmberliteXE-89 cation exchange resin on the hydrogen cycle at a rate of 6.1ml./minute (i.e. 15 minute superficial contact time).

This resin is a carboxylic acid type of cation exchange resin suppliedby the Rohm & Haas Company, of Philadelphia, Pa., having a dissociationconstant about 5 times that for the Amberlite IRC-SO cation exchangeresin supplied by the same company (also of the carboxylic acid type). 3

After the passage of the filtered broth through the column, the resinwas removed from the column and placed in a beaker and 6 N ammoniumhydroxide added to the resin in small amounts, while stirring; to adjustthe pH of the resin to from 8.5 to 9. After the pH had remained constantat 8.5 for atleast a 15 minute interval Without further addition ofammonium hydroxide, the resin was replaced in the column and washed withWater until the eluate became colorless. The following table shows theresults:

The yield across the resin column was thus 70,600 units from 84,600units introduced into the column, representing 83%. The decrease in thevolume of the bacitracin solution was from 2300 ml. to 210 ml.,represent ing the elimination of 91% of the initial volume, while thepotency of the solutionwas increased 9.2 fold.

EXAMPLE 4 One liter of bacitracin whole broth similar to that used inExample 1, was acidified to pH 3 with sulfuric acid, and after adding10% by weight of supercel as an admix, was filtered. The filter cake waswashed with 200 ml. of water, and the combined filtrate was passeddownflow through a column containing 20 ml. of Dowex 50-X1 cationexchange resin on the hydrogen cycle at a rate of 1.3 m1./minute (i.e.15 minute superficial contact time). v

This resin was available from the Dow Chemical Company, of Midland,Michigan, and wasprepared from the copolymerization of styrene withdivinylbenzene, the X1 indicating that it has a 1% cross linkage. The"resin has a sulfonic acid grouping as its functional group and is astrongly acidic cation exchange resin.

After the passage of the filtered broth through the v introduced intothe column, followed by a 1 N solution of .ammmonium hydroxide.lecteduntilthe pH of the elfluent rose to 9, constituted the eluate orrich cut. The following table shows the results Table 4 Vol.,

Assay, ml. u.

Material Iml.

Whole broth Spent broth.. Eluate The yield across the resin columnwas'thus 20,500

vunits from 37,200 units introduced into the column, representing 55%.The decrease in the volume of the 'bacitracin solution was from 940 ml.to sz-mt, reprezsenting the elimination of 91% of the initial volume,

while the potency of the solution was increased 6.3 fold.

' EXAMPLE 5 500 ml. of filtered bacitracin broth (derived from wholebroth similarly as described in Example 1), was

passed downfiow through a column containing 20 ml.

of Dowex 50-X1 cation exchange resin on the hydrogen cycle at a rate ofl mL/minute (i.e. 20 minutes super- .ficial contact time). This resin isdescribed in detailin able 5 Total Activity,

Material units 1. Filtered brotli 2. Spent broth 3. Eluate The yieldacross the resin column was thus 13,300 units from 24,400 unitsintroduced into the column, representing 55%. The decrease in the volumeof the bacitracin solution was from 500 ml. to 55 ml., representing theelimination of 89% of the initial volume, while the potency of thesolution was increased 5.0 fold.

EXAMPLE 6 2080 ml. of filtered bacitracin 'broth (derived from wholebroth similarly as described in Example 1) was passed downfiow through acolumn containing 70 ml. of Duolite C-1 0 cation exchange resin on thehydrogen cycle at a rate of 3.5 ml./minute (i.e. 20 minute superficialcontact time).

This resin was avaliable from the Chemical Process Company and wasprepared from a highly expanded phenolic matrix with omega sulfonic acidas its functional group. It is supplied in highly porous granules.

1 1 After washing the resin column similarly as described in Example 1,the activity was eluted from the resin The efiluentthat was col- 8 I bypassing 1 N ammonium hydroxide through the resin. The following tableshows the results:

Table 6 Material Vol., Assay, Total Ac- Purity,

ml. u./i.nl. tivity, units uJmg.

1. Filtered b oth 2,080 25. a as, 300 '0. s 2. Spent broth 2, 280 1. 73, 800 .3. Eluate 305 72 28,400 4. 03

The yield across the resin column was thus 28,400 units from 53,300units introduced into the column, representing 53%, while the purity ofthe active material, in terms of activity per total solid content, wasincreased from 0.8 to 4.03, representing a 5.1 fold increase. Thedecrease in the volume of the bacitracin solution was from 2080 ml. to395 ml., representing the elimination of 81% of the initial volume,while the potency of the solution was increased 2.8 fold.

EXAMPLE 7 1540 ml. of filtered bacitracin broth (derived from wholebroth similarly as described in Example 1) was 'passed downfiow througha column containing 39 m1. of

Duo lite C25 cationexchange resin having a cross linkage of around 68%.The resin was on the hydrogen cycle and the rate of flow through it was1.9 ml./minute (i.e. 20 minute superficial contact time).

This resin was available from the Chemical Process Company, and as withthe Duolite C-25 resin described in Example 1, is prepared from a matrixproduced by the copolymerization of styrene with divinylbenzene, withnuclear sulfonic acid as its functional group. The resin used in thisExample 7 difi'ered from that used in Example 1, however, in that it wasmore highly cross linked.

After washing the resin column similarly as described in Example 1, theactivity was eluted from the resin by passing ice cold 1 N ammoniumhydroxide through the resin. The following table shows the results:

The yield across the resin column was thus 8,500 units from 58,100 unitsintroduced into the column, representing 15%. The decrease in the volumeof the bacitracin solution was from 1540 ml. to 60 ml., representing'theelimination of 96% of the inital volume, while the potency of thesolution was increased 3.8 fold.

EXAMPLE 8 200 ml. of' filtered bacitracin broth (derived from wholebroth similarly as described in Example 1), was passed downfiow througha column containing 20 ml. of Amberlite 1RC50 cation exchange resin. Theresin was on the hydrogen cycle and the rate of flow through it was 1mL/minute (i.e. 20 minute superficial contact time).

This resin was a carboxylic acid type of cation exchange resin suppliedby the Rohm & Haas Company-in which the exchange groups are weaklyacidic, and which exhibits high capacity and great affinity for hydrogenl0llS.

After washing the resin column similarly as described in Example 1, theactivity was eluted from the resin by 9 passing 1 N ammonium hydroxidethrough the resin. The following table shows the results:

Table 8 Material Total Activity, units Assay,

Purity, u./rn1.

Vol.,

ml. u./mg.

1. Filtered broth 200 44 2. Spent broth"..- 216 7.4 3. Eluate 156 30SUMMARY OF EXAMPLES The results of the foregoing examples are summarizedin the following table:

10 tional groups nuclear sulfonic acids and having a cross linkage ofthe order of 1 to 2 percent, with said resin being in the hydrogen form,and eluting the adsorbed bacitracin from said res-in with a weak base.

2. The process according to claim 1 in which the pH of the broth isadjusted to about 4 before it contacts the resin.

3. The process of recovering and concentracting bacitracin elaborated bythe fermentation of B. subtilis in an aqueous solution containing about6% soy bean meal as the essential nutrient medium, which comprisesfiltering the whole broth to remove the mycelia, acidifying the filteredbroth to about pH 4, passing the filtered broth through a column of acation exchange resin of the styrene-divinylbenzene type having nuclearsulfonic acids as its functional groups and having a cross linkage ofthe order of 1 to 2% and operating in the hydrogen form, eluting theabsorbed bacitracin from said resin with dilute aqueous ammonia, andconcentrating the rich cut of the eluate by heating it under vacuum atnot over C. until the pH of the solution is less than 9.2.

Table 9 RECOVERY AND CONCENTRATION OF BACITRACIN Decreasein Yield Volumeof Approximate Type of Elu- Across Liquid Increase in Approxlmate lu-Example Cation Exchange Resin (On H Cycle) tion (With Column, (Amount ofPotency of crease 111 Pur ty Weak Base) Percent Initial Vol- Solution(Based on Solids) ume Eliminated), Percent Duolite 0-25; 2% cross linkedColumn 7 X and 8X Not determined. do do 78 14X Do. Amberlite XE-89 Batch83 91 9X DO Dowex 50-X1; 1% cross linked d0 55 91 6X 0 do 55 89 5X D0Duolite 0-10.. 53 81 3X 5X.

Duolite 0-25; about 68% cross linked 15 96 4X Not determined. AmberlitcIRC-50 53 22 1X 9X- Wh t 1 d References Cited in the file of this patenta 18 C aime 1S: 1. The process of recovering and concentrating bac-UNITED STATES PATENTS itracln from aqueous filtered fermentation brothcon- 2,528,022 Van Dolah et al. Oct. 31, 1950 taining on the order of 3%proteinaceous solids which 45 2,582,921 Charney Jan. 15, 1952 comprisesintimately contacting the broth with a syn- 2,667,441 Nager Jan. 26,1954 thetic organic cation exchange resin having as its func- 2,776,240Shortridge Jan. 1, 1957

1. THE PROCESS OF RECOVERING AND CONCENTRATING BACITRACIN FROM AQUEOUSFILTERED FERMENTATION BROTH CONTAINING ON THE ORDER OF 3% PROTEINACEOUSSOLIDS WHICH COMPRISES INTIMATELY CONTACTING THE BROTH WITH A SYN THETICORGANIC CATION EXCHANGE RESIN HAVING AS ITS FUNCTIONAL GROUPS NUCLEARSULFONIC ACIDS AND AHVING A CROSS LINKAGE OF THE ORDER OF 1 TO 2PERCENT, WITH SAID RESIN BEING IN THE HYDROGEN FROM, AND ELUTING THEADSORBED BACITRACIN FROM SAID RESIN WITH A WEAK BASE.